Drug-Loaded Microparticles
Multiple types of cancer originate from organs located within the peritoneal cavity, e.g., pancreatic, liver, colorectal, and ovarian cancer. The peritoneal cavity also is a site for metastasis of cancer originating from organs outside of the peritoneal cavity during the late stage of disease, e.g., lung cancer. Within the peritoneal cavity, tumors can be found in pelvic and abdominal peritoneal surfaces, other peritoneal organs, e.g., intestinal mesenteries, bladder, omentum, diaphragm, lymph nodes and liver. Obstruction of the diaphragmatic or abdominal lymphatic drainage by tumor cells leads to decreased outflow of peritoneal fluid resulting in carcinomatosis or ascites.
Intraperitoneal chemotherapy, where the drug is directly instilled into the peritoneal cavity, has been used to treat peritoneal cancer, e.g., advanced ovarian and colon cancer (Otto, S. E., J Intraven. Nurs., 18:170-176, 1995; Collins, J. M., J Clin Oncol, 2:498-504, 1984). Intraperitoneal administration of platinated compounds, e.g., cisplatin, and Taxol®, the commercially available formulation of paclitaxel, has produced some benefits in patients and extended the survival time by about 20% (Gadducci, et al., Gynecol Oncol 76:157-162, 2000; Markman, et al. J Clin Oncol, 19:1001-1007, 2001). However, intraperitoneal chemotherapy has the following drawbacks that limit its use. Intraperitoneal treatments are usually administered through indwelling catheters, every 3 weeks for 6 treatments. The two major side effects are infection associated with the prolonged use of a catheter and abdominal pain due to the presentation of high drug concentrations in the peritoneal cavity (Francis, et al., J Clin Oncol, 13:2961-2967, 1995). Further, intraperitoneal administration requires hospitalization and is associated with substantial costs. These reasons have contributed to the reluctance of the medical community to use intraperitoneal treatments in spite of its demonstrated survival benefits. The current invention overcomes these various deficiencies.
In an earlier invention (U.S. patent application Ser. No. 09/547,825), Applicants showed that when anticancer drugs (e.g., paclitaxel, doxorubicin) are administered to the exterior of a solid tumor, as would be the case during regional intraperitoneal therapy, the drug penetration into solid tumors is very slow and limited to the tumor periphery. Applicants further disclosed a method to overcome this penetration problem. This method consists of using an apoptosis induction treatment (e.g., treatment with paclitaxel or doxorubicin) to expand the interstitial space within a tumor and thereby improve the penetration and distribution throughout solid tumors of the concurrently and subsequently administered drugs. This method is referred to as the “Tumor Priming” method in the current application, and has two requirements. The first requirement is that the drug concentrations must be sufficient to induce apoptosis. Applicants have shown that treatment with 200 nM paclitaxel for 3 hours is sufficient to induce apoptosis in multiple human tumor cells (Au, et al., Cancer Res., 58:2141-2148, 1998). The second requirement is that the time interval between the apoptosis-inducing pretreatment and subsequent treatments must be sufficient to allow apoptosis to occur, e.g., 16-24 hours for paclitaxel (Au, et al., Cancer Res., 1998; Jang, et al., J. Pharmacol. Exper. Therap., 296:1035-1042, 2001). These requirements are captured in the current invention.
Applicants further discovered that after intraperitoneal administration, the commercial Taxol® formulation was rapidly cleared from the peritoneal cavity, due to rapid absorption through the peritoneum, a thin membrane lining the peritoneal cavity, and due to drainage through the lymphatic system (see Examples 4 and 6, infra).
Based on these various considerations and discoveries, Applicants arrived at the conclusion that therapeutically useful intraperitoneal chemotherapy can be accomplished, if the treatment satisfies some or most of the following desired properties. First, the drug should have activity against the intended target cancer type. Second, the treatment should be easy to administer, does not require the use of indwelling catheter over long period of time, e.g., longer than one day or two days, and does not require frequent administration, e.g., no more than once a week. Third, the rate of drug presentation should be optimized so that the drug amount and, therefore, the drug concentration in the peritoneal cavity is high enough to provide adequate control of the disease but at the same time low enough to not produce significant local toxicity. Fourth, the drug or drug formulation must be able to penetrate into and widely distribute within solid tumors. Fifth, the drug or drug formulation should have a long retention in the peritoneal cavity where the tumors are located. Sixth, the drug or drug formulation should have high affinity to tumor cells and localize on tumor surface or within the tumor mass. Seventh, the distribution of the drug or drug formulation should be similar to the distribution or dissemination of tumor cells, within the peritoneal cavity or organs. Most or all of these desired features are captured in the current invention, i.e., drug-loaded microparticles.
The drug-loaded microparticles are designed to release the drug at two rates; a rapid release to provide sufficiently high drug concentration to induce apoptosis and a slower release to provide sustained drug delivery to tumors. The apoptosis induction promotes the penetration and distribution of the remaining microparticles and the remaining drug subsequently released from the slow-release particles. The slow drug release over a long time period, e.g., days, weeks or months, offers the patient the convenience of a single administration, reduces the frequency of treatments, eliminates the need of hospitalization, reduces the health care costs, eliminates the need of using indwelling intraperitoneal catheters and thereby reduces the risk of infections and improves the quality of life for a patient, and reduces the local toxicity due to the high local concentrations in the peritoneal cavity resulting from rapid bolus presentation of the entire dose all at once. These particles, due to their sizes and properties, are retained in the peritoneal cavity, adhere to tumors, and show similar distribution within the peritoneal cavity or organs.
The particles also can be combinations of two or more types of particles, with at least one type releasing the drug rapidly to induce apoptosis while the remaining types release the drug more slowly. Examples of microparticles loaded with a widely used anticancer drug paclitaxel are provided to demonstrate the utility of these particles, with respect to producing superior tumor targeting and antitumor activity in mice bearing peritoneal tumors, as compared to the commercial paclitaxel formulation, i.e., Taxol®, where paclitaxel is solublilized in Cremophor and ethanol.
Applicants further disclose that other drugs or agents can be formulated in the same microparticles for the purpose of treating peritoneal cancer.
Applicants further describe that drug-loaded particles can be used to treat tumors located in organs or regions that are readily accessible by local or regional administration.
The current invention uses biodegradable particles made of gelatin and PLGA polymers, loaded with agents of therapeutic utility. One of the agents formulated in the gelatin and PLGA particles is paclitaxel. A variety of biodegradable polymer bound paclitaxel formulations have been developed and have been shown to inhibit tumor growth and angiogenesis in animal models with minimal systemic toxicity; however, the release kinetics of paclitaxel in these systems, which range from about 10-25% of the drug released in approximately 50 days, are, most likely, not optimal for clinical use (U.S. Pat. No. 6,447,796). In addition, the purpose of most of these earlier studies was to achieve systemic, rather than regional, delivery of the drug. The advantages of biodegradable polymers as a carrier for agents of therapeutic utility are art-recognized (U.S. Pat. No. 6,447,796), and include: (1) complete biodegradation, requiring no follow-up surgery to remove the drug carrier when the drug supply is exhausted; (2) tissue biocompatibility; (3) ease of administration; (4) controlled, sustained release of the encapsulated drug upon hydrolysis of the polymer; (5) in case of regional use, minimization or elimination of systemic toxicity, such as neutropenia; and (6) the convenience of the biodegradable polymer system itself, in terms of versatility. Many of these advantages, similarly, apply to gelatin drug release particles.
Paclitaxel-Loaded Nanoparticles for Intravesical Therapy of Bladder Cancer
Intravesical chemotherapy is used to reduce bladder cancer recurrence and/or progression (Kurth, K. H., Semin. Urol. Oncol., 14: 30-35, 1996). Intravesical chemotherapy provides the advantage of selectively delivering drugs in high concentration to the tumor-bearing bladder, while minimizing the systemic exposure. Applicants have shown that treatment failure in superficial bladder cancer patients to chemotherapy, e.g., mitomycin C, e.g., doxorubicin, is in part due to the low drug delivery to tumors located in the bladder tissue (Dalton, et al., Cancer Res., 51: 5144-5152, 1991; Schmittgen, et al., Cancer Res., 51: 3849-3856, 1991; Wientjes, et al., Pharm. Res., 8: 168-173, 1991; Wientjes, et al., Cancer Res., 51: 4347-4354, 1991; Wientjes, et al., Cancer Res., 53: 3314-3320, 1993; Chai, et al., J Urol., 152: 374-378, 1994; Wientjes, et al., Cancer Chemother Pharmacol. 37: 539-546, 1996; Au, et al., J. Natl. Cancer Inst., 93: 597-604, 2001; U.S. Pat. No. 6,286,513 B1). The low drug delivery, in turn, was due to several reasons. First, only a fraction of the mitomycin C or doxorubicin dose, e.g., ˜3% to ˜5%, was able to penetrate the urothelium that lines the inner surface of the bladder cavity. Second, the concentration of the drug was diluted by the presence of residual urine when the drug was administered and by the urine produced during the treatment interval, e.g., 2 hours. Third, the total exposure of tumor cells to the drug, e.g., mitomycin C, was restricted to the length of the treatment interval due to the patient's need to empty his or her bladder. Fourth, the residence of the drug, e.g., mitomycin C, in the bladder tissue is dictated by the treatment duration and is largely terminated within minutes after the patient empties his or her bladder.
Based on these various considerations and discoveries, Applicants arrived at the conclusion that therapeutically useful intravesical chemotherapy can be accomplished, if the treatment satisfies some or most of the following desired properties. First, the drug should have activity against bladder cancer. Second, a large fraction of the drug present in the urine must be able to penetrate the urothelium. Third, the residence time of the drug in the bladder tissues should exceed the duration of the treatment. Fourth, the drug concentration in the urine should be independent of the urine volume. Paclitaxel is active against bladder cancer (Roth, B., J. Semin. Oncol., 22: 1-5, 1995) and, as shown by Applicants, readily partitioned across the urothelium, e.g., 50% of the dose (Song, et al., Cancer Chemother. Pharmacol., 40: 285-292, 1997). Applicants further discovered that paclitaxel is retained in tumor cells after removing drug from the extracellular matrix (Kuh, et al., J. Pharmacol. Exp. Ther., 293: 761-770, 2000), a property that offers the opportunity of extending drug action beyond the 2 hour treatment duration. However, the commercial paclitaxel formulation, e.g., Taxol®, is not useful, because the Cremophor used to solubilize paclitaxel, by entrapping paclitaxel in micelles, reduces the free fraction of paclitaxel and consequently lowers the drug penetration into the bladder tissue (Knemeyer, et al., Cancer Chemother. Pharmacol., 44: 241-248, 1999).
Applicants, therefore, invented paclitaxel-loaded nanoparticles that satisfy all of the desired properties identified in Applicants' discoveries. These nanoparticles release a significant fraction of the drug load within the 2-hour treatment interval, such that the paclitaxel concentration in the tissue is sufficient to produce antitumor activity in human bladder tumors (e.g., Example 8). The amount of paclitaxel released from the nanoparticles is limited by the drug solubility in the urine. Hence, the drug concentration in the urine remains relatively constant and is independent of the urine volume (e.g., Example 10). The paclitaxel released from the nanoparticles and penetrated into the bladder also is retained in the bladder tissues for periods extending beyond the 2-hour treatment duration (e.g., Example 8). Finally, Applicants determined that the nanoparticles were effective against naturally occurring bladder cancer in pet dogs (e.g., Example 10).
Applicants also disclose a method to modify the drug-loaded nanoparticles to prolong the retention of these particles in the bladder cavity or bladder tissues beyond the treatment duration, e.g., by coating the gelatin framework with bioadhesive molecules (e.g., Example 7).
Applicants further disclose that other lipophilic compounds can be formulated in the same gelatin nanoparticles.
Finally, Applicants disclose that intravenous administration of gelatin nanoparticles resulted in increased localization of the drug contents in the kidney (e.g., Example 11). Hence, methods and compositions for selective drug delivery to the kidney are also provided.
Definitions
In order to provide a clear and consistent understanding of the invention, certain terms employed in the specification, examples, and the claims are, for convenience, collected here.
As used herein, the term “aberrant growth” refers to a cell phenotype, which differs from the normal phenotype of the cell, particularly those associated either directly or indirectly with a disease such as cancer.
As used herein, the term “administering” refers to the introduction of an agent to a cell, e.g., in vitro, a cell in a mammal, i.e., in vivo, or a cell later placed back in the animal (i.e., ex vivo).
As used herein, the terms “agent”, “drug”, “compound”, “anticancer agent”, “chemotherapeutic”, “antineoplastic”, and “antitumor agent” are used interchangeably and refer to agent(s) (unless further qualified) that have the property of inhibiting or reducing aberrant cell growth, e.g., a cancer. The foregoing terms are also intended to include cytotoxic, cytocidal, or cytostatic agents. The term “agent” includes small molecules, macromolecules (e.g., peptides, proteins, antibodies, or antibody fragments), and nucleic acids (e.g., gene therapy constructs), recombinant viruses, nucleic acid fragments (including, e.g., synthetic nucleic acid fragments).
As used herein, the term “apoptosis” refers to any non-necrotic, well-regulated form of cell death, as defined by criteria well established in the art.
As used herein, the terms “benign”, “premalignant”, and “malignant” are to be given their art recognized meanings.
As used herein, the terms “cancer”, “tumor cell”, “tumor”, “leukemia”, or “leukemic cell” are used interchangeably and refer to any neoplasm (“new growth”), such as a carcinoma (derived from epithelial cells), adenocarcinoma (derived from glandular tissue), sarcoma (derived from connective tissue), lymphoma (derived from lymph tissue), or cancer of the blood (e.g., leukemia or erythroleukemia). The term cancer or tumor cell is also intended to encompass cancerous tissue or a tumor mass which shall be construed as a compilation of cancer cells or tumor cells. In addition, the term cancer or tumor cell is intended to encompass cancers or cells that may be either benign, premalignant, or malignant. Typically a cancer or tumor cell exhibits various art recognized hallmarks such as, e.g., growth factor independence, lack of cell/cell contact growth inhibition, and/or abnormal karyotype. By contrast, a normal cell typically can only be passaged in culture for a finite number of passages and/or exhibits various art recognized hallmarks attributed to normal cells (e.g., growth factor dependence, contact inhibition, and/or a normal karyotype).
As used herein, the term “cell” includes any eukaryotic cell, such as somatic or germ line mammalian cells, or cell lines, e.g., HeLa cells (human), NIH3T3 cells (murine), embryonic stem cells, and cell types such as hematopoietic stem cells, myoblasts, hepatocytes, lymphocytes, and epithelial cells and, e.g., the cell lines described herein.
As used herein, the terms “peritoneal”, “intraperitoneal”, “peritoneally”, or “intraperitoneally” are used interchangeably, and are related to the peritoneal or abdominal cavity.
As used herein, the terms “locally”, “regionally”, “systemically” refer to, respectively, the administration of a therapy “locally”, e.g., into a tumor mass, “regionally”, e.g., in a general tumor field or area suspected to be seeded with metastases, or “systemically” e.g., orally or intravenously with the intent that the agent will be widely disseminated throughout the subject.
As used herein, the term “pharmaceutically acceptable carrier” is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals.
As used herein, the term “pharmaceutical composition” includes preparations suitable for administration to mammals, e.g., humans. When the compounds of the present invention are administered as pharmaceuticals to mammals, e.g., humans, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient (e.g., a therapeutically-effective amount) in combination with a pharmaceutically acceptable carrier.
As used herein, the term “subject” is intended to include human and non-human animals (e.g., mice, rats, rabbits, cats, dogs, livestock, and primates). Preferred human animals include a human patient having a disorder characterized by aberrant cell growth, e.g., a cancer.
As used herein, the term “microparticles” refers to particles of about 0.1 μm to about 100 μm about 0.5 μm to about 50 μm, 0.5 μm to about 20 μm in size, advantageously, particles of about 1 μm to about 10 μm in size, about 5 μm in size, or mixtures thereof. The microparticles may comprise macromolecules, gene therapy constructs, or chemotherapeutic agents, for example. Typically microparticles can be administered locally or regionally, for example.
As used herein, the term “nanoparticles” refers to particles of about 0.1 nm to about 1 μm, 1 nm to about 1 μm, about 10 nm to about 1 μm, about 50 nm to about 1 μm, about 100 nm to about 1 μm, about 250-900 nm in size, or, advantageously, about 600-800 nm. The nanoparticles may comprise macromolecules, gene therapy constructs, or chemotherapeutic agents, for example. Typically, nanoparticles can be administered to a patient via local, regional, or systemic administration.
As used herein, the term “particles” refers to nanoparticles, microparticles, or both nanoparticles and microparticles.
As used herein, the term “formulation” refers to the art-recognized composition where a therapeutically active agent is incorporated in a dosage form.
As used herein, the terms “fast-release formulation” and “rapid-release formulation” refer to a formulation of a drug which releases preferably >10%, more preferably >20%, more preferably >30%, more preferably >40%, more preferably >50%, and even more preferably >60% of its drug contents within one day. Examples of fast-release formulations include microparticle and nanoparticle formulations. Methods used to prepare these formulations are described in Examples 3 and 7.
As used herein, the term “slow-release formulation” refers to a formulation of a drug wherein the drug is delivered to a site of interest for a sustained period of time and includes formulations which maintain release of its drug contents preferably for several days, more preferably several weeks or longer.
As used herein, the term “tumor priming method” refers to a method of enhancing the penetration of a therapeutic drug by “priming” the tumor with an apoptosis inducing agent to decrease tumor cell density and increase therapeutic agent accessibility to the tumor mass. This method involves the use of an apoptosis inducing agent, such as paclitaxel, in doses and for periods of time sufficient to cause apoptosis in the tissue to thereby allow for enhanced penetration of the tumor therapeutic agent (or simply “therapeutic agent”) into the tissue (e.g., by creating channels within the tissues). Thus, the apoptosis inducing agent is used as a pretreatment before the therapeutic dose of the therapeutic agent is delivered to the tissue, and this pretreatment allows for enhanced penetration of the therapeutic agent into the tissue as compared to when the pretreatment is not used. The apoptosis inducing agent may also have therapeutic activity and thus may also be used as the therapeutic agent (i.e., the same drug may be used as the apoptosis inducing agent and the therapeutic agent). Alternatively, the apoptosis inducing agent may be used to enhance delivery of other types of drugs or vehicles for treatment into tissues (i.e., the apoptosis inducing agent and the therapeutic agent may be different drugs, or the therapeutic agent may be contained in nanoparticles or microparticles, where the delivery of the nanoparticles or microparticles to the tumor tissue is enhanced, compared to when the pretreatment is not used).
As used herein, the term “PLGA”, or “poly(lactide-co-glycolide)” refers to a copolymer consisting of various ratios of lactic acid or lactide (LA) and glycolic acid or glycolide (GA). The copolymer can have different average chain lengths, resulting in different internal viscosities and differences in polymer properties. PLGA is used for the preparation of microparticles or nanoparticles, usually containing therapeutic agents. Methods used to prepare these particles are described in Example 3.
As used herein, the term “gelatin” refers to a denatured form of the connective tissue protein collagen. Gelatin aggregates, formed in solution, are stabilized by cross-linking the protein chains. Using the preparation method of Example 7, gelatin forms gelatin nanoparticles, usually loaded with paclitaxel. Gelatin is available in different protein chain lengths, indicated by different Bloom numbers. Larger Bloom numbers indicate longer chain lengths.
As used herein, the term “IC50” refers to the drug concentration or dose that results in 50% of the drug effect.
As used herein, the term “burst release” refers to the art-recognized definition of the initial, rapid release of a fraction of the drug load from a formulation, which is typically followed by a slower release of the remainder of the drug load.
As used herein, the terms “localize” and “concentrate” are used interchangeably, to indicate the preferential distribution at a specific site, e.g., tumor tissues.
As used herein the term “bioadhesive” means natural, synthetic or semi-synthetic substances that adhere and preferably strongly adhere to a surface such as skin, mucous membrane, and tumor. Suitable bioadhesives include poly(lysine), fibrinogen, those prepared from partially esterified polyacrylic acid polymers, including polyacrylic acid polymers, natural or synthetic polysaccharides such as cellulose derivatives including methylcellulose, cellulose acetate, carboxymethylcellulose, hydroxyethylcellulose, pectin, and a mixture of sulfated sucrose and aluminum hydroxide.
As used herein, the term “interstitial cystitis” refers to the art-recognized medical condition of chronic, painful inflammatory condition of the bladder wall. As used herein, the terms “biodegradable” or “bioerodible” polymer refer to polymers that can degrade into low molecular weight compounds, which are known to be involved normally in metabolic pathways. The terms also include polymer systems which can be attacked in the biological milieu so that the integrity of the system, and in some cases of the macromolecules themselves is affected and gives fragments or other degradation by-products which can move away from their site of action, but not necessarily from the body.
Broad Statement of the Invention
A composition for delivering a tumor therapeutic agent to a patient, which includes a fast-release formulation of a tumor apoptosis inducing agent, a slow-release formulation of a tumor therapeutic agent, and a pharmaceutically acceptable carrier. An apoptosis inducing agent in a pharmaceutically acceptable carrier may be administered before or concomitantly therewith. Nanoparticles or microparticles (e.g., cross-linked gelatin) of the therapeutic agent (e.g., paclitaxel) also may be used. The nanoparticles or microparticles may be coated with a bioadhesive coating. Microspheres that aggregate to block the entrance of the lymphatic ducts of the bladder to retard clearance of the microparticles through the lymphatic system also may be employed.
This invention also uses drug-loaded gelatin and poly(lactide-co-glycolide) (PLGA) nanoparticles and microparticles to target drug delivery to tumors in the peritoneal cavity, bladder tissues, and kidneys.